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At least 253 records · Page 14Linked to original sources
PubMed2002

High-resolution electrophysiological assessment of human embryonic stem cell-derived cardiomyocytes: a novel in vitro model for the study of conduction.

The goal of the present report was to establish a new in vitro model for the study of impulse propagation in human cardiac tissue. By using the human embryonic stem cell differentiating system, spontaneously contracting areas were generated in three-dimensional differentiating cell aggregates (embryoid bodies). Morphological analysis revealed an isotropic tissue of early-stage cardiac phenotype. Gap junctions, assessed by immunostaining of connexin43 and connexin45, were distributed along the cell borders. High-resolution activation maps demonstrated the presence of a functional syncytium with stable focal activation and conduction properties. Conduction was significantly slower in narrow bands of contracting tissue compared with broad cardiomyocyte regions. Establishment of this unique in vitro human model may be used for the assessment of long-term structure-function relationships, for pharmacological studies, for tissue engineering, and may permit the study of genetically modified cardiomyocytes.

Action Potentials↗
PubMed1989

Round window membrane permeability. An in vitro model.

The round window membrane of the mongolian gerbil was dissected out, together with its bony niche. The preparation was mounted between two glass chambers representing the middle ear cavity and the perilymphatic space respectively. Passage through the round window membrane did not occur within 3 h when testing high-density lipoprotein with a molecular weight 115-350 kD. Horse-radish peroxidase, with a molecular weight of 48 kD, passed the round window membrane at a mean rate of 6 micrograms/h when the concentration of the protein was 10 g/l in the middle ear chamber. The present in vitro model is considered to be free from leakage artifacts between the chambers. Passage rates for different substances through the round window membrane can be calculated under controlled conditions using this type of in vitro model.

Albumins↗
PubMed1996

An in vitro Model for Studying Mechanisms Underlying Synoviocyte-Mediated Cartilage Invasion in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease of joints involving the pathological development of an invasive and destructive pannus tissue which contributes to the loss of cartilage and bone. To further analyze the process of cartilage degradation and invasion, we have developed an in vitro model composed of cartilage matrix and synoviocytes (isolated from RA pannus tissue, as well as normal synovial membrane). The matrix is derived from pig articular cartilage and contains collagen type II and proteoglycans and is similar in composition to human cartilage. Data generated from this model reveal that synoviocytes isolated from RA pannus tissue invaded cartilage matrix in a manner which directly correlated with the severity of the disease. Analysis of mechanisms associated with the invasive process demonstrate that highly invasive RA synoviocytes maintain a round morphology during attachment and spreading on cartilage matrix, compared with their normal counterparts. Furthermore, the level of secretion of matrix metalloproteinase (MMP) activity was shown to correlate with the RA phenotype, which could be modulated with a novel MMP inhibitor. Normal synoviocytes could be "converted" to an RA phenotype by specific inflammatory cytokines, such that invasion of cartilage matrix was augmented by culturing these cells in the presence of 5 U/ml IL-1b or 18 U/ml TGFb. Invasion was inhibited by 150 U/ml TNFa, and unaffected by 100 ng/ml PDGF. In addition, synovial fluid from RA patients induced invasion of normal synoviocytes, in a concentration dependent manner, from 150% to 460%; however, synovial fluid from another inflammatory arthritidy (Crohn's) did not augment invasion to the same degree. Moreover, this "conversion effect" appears to be specific for synoviocytes, since similar effects could not be achieved with human skin fibroblasts. This in vitro model of synoviocyte-mediated cartilage invasion allows for further molecular characterization of the invasive properties of the synoviocyte which contribute to RA.

Journal Article↗
PubMed2006

Hypoxia induced chemokine expression in nasal epithelial cells: development of an in vitro model for chronic rhinosinusitis.

Chronic rhinosinusitis (CRS) is defined as an inflammatory condition involving the paranasal sinuses and the lining of the nasal passages that persists longer than 12 weeks. It is one of the most common chronic diseases today, affecting up to 15% of the adult population in the Western world with a dramatic increase in prevalence. One hallmark of chronic inflammation in CRS is the predominance of eosinophils and T lymphocytes in the inflamed tissue. We pursued the hypothesis that the blockage of the paranasal sinuses induces hypoxic conditions, which subsequently lead to the induction of chemotactic activity, attracting inflammatory cells. To this end, we established an in vitro model by showing that hypoxia is able to induce the release of chemokines in nasal epithelial cells. Furthermore, we show that this induction leads to the migration of eosinophils and neutrophils. Finally, we demonstrated the applicability of this in vitro model by showing its sensitivity to the glucocorticoid dexamethasone, which is used in the clinical situation. These results suggest that this nasal epithelial cell culture model may allow the evaluation of novel anti-inflammatory compounds for the treatment of CRS directly on the relevant target cells in vitro. This approach may result in replacing and refining animal experimentation in the screening of new therapeutics for CRS.

Cells, Cultured↗
PubMedDate not supplied

Development and evaluation of an in vitro model for the analysis of cigarette smoke effects on cultured cells and tissues.

INTRODUCTION: Smokers have an increased risk for a variety of diseases. Among the most prominent is atherosclerosis, the leading cause of death in the Western world. Although this conjunction is accepted knowledge, the basic biological mechanisms and the identities of the active tobacco smoke constituents surprisingly are still unknown. One reason for this is the lack of accurate in vitro models. METHODS: Cell culture experiments, including cell morphology and cell death analyses, high-performance liquid chromatography, and liquid chromatography coupled to mass spectrometry via an electrospray ionization interface allowing collision-induced dissociation analyses, were applied. RESULTS AND DISCUSSION: In this study, we present and validate an in vitro model that has proven to be useful for standardized studies of cellular and histological effects of cigarette smoke. The system consists of a cigarette smoke sampling device in which water-soluble cigarette smoke constituents pass over from the gas phase into the aqueous phase resulting in nicotine concentrations identical to the in vivo concentrations, suggesting in vivo similar conditions for gas-to-liquid compound exchange.

Cell Survival↗
PubMed1996

An in Vitro Model of Oligodendrocyte Destruction by Nitric Oxide and Its Relevance to Multiple Sclerosis

There is mounting evidence that nitric oxide (NO) is produced in the brains of patients with multiple sclerosis (MS) and in the experimental model of MS, experimental autoimmune encephalomyelitis, after the induction of Type II nitric oxide synthase (iNOS). Because NO can cause a variety of biological insults that compromise or even kill normal cells, we studied the effects of NO on oligodendrocytes since they are a target in MS tissue. In an in vitro model, we have been able to demonstrate that NO causes damage to oligodendrocytes preferentially, sparing microglia almost completely and affecting some but not all astrocytic functions. This article describes the types of assays used to measure morphological changes, mitochondrial dysfunction, DNA strand breaks, and cell death brought on by NO or peroxynitrite (ONOO-) as well as a comprehensive review of the various techniques and sensitivities of NO and iNOS assays that would be applicable to similar in vitro models.

Journal Article↗
PubMed2006

Effect of photodynamic therapy on the function of the outer blood-retinal barrier in an in vitro model.

BACKGROUND: Photodynamic therapy (PDT) is a well established clinical treatment for age-related macular degeneration (AMD), and comprises intravenous injection of verteporfin and subsequent application of a non-thermal laser beam to the area of AMD to induce selective vascular occlusion. Since there is evidence that PDT may cause outer blood-retinal barrier (BRB) breakdown and possibly RPE cell alteration, we investigated the effect of PDT on the BRB function of the RPE in an in vitro model. METHODS: Twenty-one monolayers of human RPE cells were cultured on semipermeable membranes until a stable barrier function was achieved as determined by transepithelial electrical resistance (TER) and sodium fluorescein permeability. To test the effect of PDT on the outer BRB function, non-thermal laser (692 nm), verteporfin or a combination of both were applied. TER assessment prior to and after PDT was utilized to identify changes in barrier function of the RPE in this in vitro model. Finally, monolayers of RPE cells were evaluated by transmission electron microscopy (TEM). RESULTS: No significant TER decrease was observed after application of non-thermal laser alone or after administration of verteporfin in therapeutic concentrations, but combination of these modalities resulted in significantly decreased TER within 4 h. Except for intercellular blisters, no damage to the RPE was evident in TEM. Verteporfin added at concentrations higher than therapeutic doses (2 mg/ml) resulted in an immediate decrease in TER and damage to the RPE cells. CONCLUSION: The combination of a therapeutic concentration of verteporfin and application of non-thermal laser resulted in a morphologically and functionally detectable breakdown of the outer BRB function of the RPE without any damage to the RPE cells themselves in vitro. However, increasing the concentration of verteporfin can result in RPE cell damage.

Blood-Retinal Barrier↗
PubMed2003

Unsteady flow through in-vitro models of the glottis.

The unsteady two-dimensional flow through fixed rigid in vitro models of the glottis is studied in some detail to validate a more accurate model based on the prediction of boundary-layer separation. The study is restricted to the flow phenomena occurring within the glottis and does not include effects of vocal-fold movement on the flow. Pressure measurements have been carried out for a transient flow through a rigid scale model of the glottis. The rigid model with a fixed geometry driven by an unsteady pressure is used in order to achieve a high accuracy in the specification of the geometry of the glottis. The experimental study is focused on flow phenomena as they might occur in the glottis, such as the asymmetry of the flow due to the Coanda effect and the transition to turbulent flow. It was found that both effects need a relatively long time to establish themselves and are therefore unlikely to occur during the production of normal voiced speech when the glottis closes completely during part of the oscillation cycle. It is shown that when the flow is still laminar and symmetric the prediction of the boundary-layer model and the measurement of the pressure drop from the throat of the glottis to the exit of the glottis agree within 40%. Results of the boundary-layer model are compared with a two-dimensional vortex-blob method for viscous flow. The difference between the results of the simpiflied boundary-layer model and the experimental results is explained by an additional pressure difference between the separation point and the far field within the jet downstream of the separation point. The influence of the movement of the vocal folds on our conclusions is still unclear.

Air Pressure↗
PubMed1997

Cellular aspects of HIV-1 infection of macrophages leading to neuronal dysfunction in in vitro models for HIV-1 encephalitis.

HIV-1 is a hematogenously spread virus that most likely gains entry into the brain within blood-derived macrophages. Indeed, productive viral replication selectively occurs within perivascular and parenchymal blood-derived macrophages and microglia and HIV-infected macrophages have increased potential to bind and transmigrate through the blood-brain barrier. Once inside the brain, HIV-infected macrophages secrete a variety of pro-inflammatory mediators that display neuromodulatory and neurotoxic activities in several in vitro models for HIV-1 encephalitis. The final outcome regarding neuronal function and cell loss is regulated through intercellular interactions between these virus-infected cells and astrocytes. In this regard, both HIV-induced intracellular events in macrophages and interactions between HIV-infected macrophages and brain cells are reviewed as factors that might lead to neuronal injury in in vitro model systems for HIV-1 encephalitis.

AIDS Dementia Complex↗
PubMed2006

Effects of the pesticide clorpyrifos on an in vitro model of intestinal barrier.

Clorpyrifos (CPF), one of the most widely used organophosphorothionate pesticide can be detected as residues in food and drinking water; therefore the oral route is the major route of exposure for the general population, including children, following household use of this insecticide. Aim of this work was to investigate the possible acute cytotoxic effects of CPF on intestine and the integrity of the epithelial barrier, using Caco-2/TC7 cells as intestinal in vitro model. High level of CPF found inside the cells, corresponding to about 80% of the nominal concentration tested (30, 50 and 250microM), chosen as representative of the concentrations attainable in the intestinal lumen after actual levels of human oral exposure. In these conditions, no cytotoxicity in terms of cellular viability was observed. However, at the highest CPF nominal concentration (250microM) the impairment of barrier integrity was evidenced, due only to the parent compound, since no CPF metabolites could be detected in our experimental conditions. CPF itself was demonstrated to interfere with the tight junction on this in vitro model of epithelial intestinal cells, altering the barrier integrity and very likely the absorption of other co-administered chemicals.

Caco-2 Cells↗
PubMed1998

Monoacylation of ribonuclease A enables its transport across an in vitro model of the blood-brain barrier.

A major challenge in correcting disorders affecting the central nervous system is to induce blood-brain barrier (BBB) crossing of exogenous biological compounds such as proteins or specific nucleic acid sequences. Fatty acids, due to their high membrane affinity and low toxicity, are good potential candidates to promote this barrier crossing when covalently bound to proteins. In this paper, we report that regiospecific monoacylation of ribonuclease A (RNase A) enables its transport across an in vitro model of the BBB. Myristoylated, palmitoylated and stearoylated RNases A were prepared using reversed micelles as microreactors. All the purified acylated RNases A kept their original enzymatic activity. A single fatty acid moiety was linked to RNase A through the alpha-amino group of its N-terminal lysine as shown by powerful analytical techniques. The ability of monoacylated RNases A to cross an in vitro model of the BBB is strictly dependent on the acyl chain length, which must be at least 16 carbon atoms long.

Acetylation↗
PubMed1987

In vitro models of lung toxicity.

In vitro assays that emphasize cellular components critical to the host defense system have been developed to evaluate pulmonary toxicity and define deleterious changes in parenchymal cell populations. Assays that employ pulmonary alveolar macrophages (PAM) have demonstrated good correlation between macrophage toxicity and pulmonary fibrogenicity for many inorganic compounds. The PAM assays provide simple and inexpensive screens of potential respiratory tract toxicity. Many investigators screen chemicals for their ability to alter the mucosal epithelial cell conducting airways by performing tracheal organ cultures. The tracheal assays have also provided useful screens for Vitamin A analogues required for epithelial cell differentiation. Most recently, in vitro respiratory tract models have been extended to include whole-lung explants, an approach that allows for development of fibrosis and epithelial cell toxicity after in vitro exposure to inorganic and organic fibrogens. The whole-lung explant system appears to duplicate the in vivo response to a variety of lung toxins, including bleomycin, silica, and crocidolite asbestos. Together, these assays provide a description of potential toxicity to key components of the lung, emphasizing the pulmonary macrophage, conducting airways, and alveolar septae. It is expected that continued research in these models will enhance their predictive abilities and utility in risk assessment.

Animals↗
PubMed2000

In vitro model for frontal sinus obliteration with bioactive glass S53P4.

An in vitro model was used to investigate the behavior of a massive frontal sinus obliteration with bioactive glass S53P4 (BG) for clinical purposes. Two sizes of granules (0.63-0.8 mm or 0.8-1.0 mm) in 16 separate BG amounts, weight 25 g, were tested both in simulated body fluid (SBF) and a buffer containing trishydroxymethyl aminomethane citric acid (TRIS-c.a) in standard conditions. The dissolution of silicon (Si) and phosphate (P) was detected with direct current plasma atom emission spectroscopy (DCP-AES) monthly up to 6 months. The BG masses were scanned by computer tomography (CT) and the scans analyzed by Region of Interest (ROI) technique. Calcium phosphate (CaP)- and silica (Si)-gel-layers were studied by scanning electron microscopy (SEM) at 1, 3, and 6 months. Cumulative loss of Si and P was stronger in TRIS -c.a than in SBF (p < 0.0001), and it was higher with smaller than with larger granules in both solutions (p < 0.0001). This was shown correspondingly by the decrease in Hounsfield units (HU) by ROI analysis (p < 0.0001). In SBF-soaked BG masses, the CaP-layer occurred on the uppermost granules, and in TRIS-c.a at 3-6 months, on the granules in the center and lower parts. The decrease of HU seems to reveal indirectly the resorption of BG.

Body Fluids↗
PubMed2000

Cultured stromal cells: an in vitro model of prostatic mesenchymal biology.

BACKGROUND: Initial efforts to develop in vitro models to study prostatic biology focused on the culture and characterization of epithelial cells. Recently, attention has turned towards inclusion of stromal cells in experimental systems. METHODS: Improved methods to isolate and culture stromal cells have been developed. An array of markers are employed to characterize subtypes of stromal cells, with particular interest in smooth muscle differentiation. RESULTS: Defined, serum-free media are available for certain experimental applications. Conditions that promote smooth muscle differentiation have been identified. Investigators have characterized hormonal and peptide factors that regulate the growth of prostatic stromal cells, and have also described paracrine factors produced by stromal cells that influence epithelial biology. CONCLUSIONS: Prostatic stromal-cell cultures are now widely employed by a large number of investigators for a diverse array of experimental purposes. While further refinement is required to obtain model systems that fully mimic in vivo processes, the availability of stromal- and epithelial-cell cultures provides a valuable resource for studying normal prostatic biology as well as benign prostatic hyperplasia (BPH) and cancer.

Adenocarcinoma↗
PubMed1994

Dorsal root ganglia cocultured with macrophages: an in vitro model to study experimental demyelination.

The present investigation introduces an in vitro model to study macrophage properties during demyelination. Rat dorsal root ganglia (DRG) were cultured for obtaining myelinated peripheral nerve fibers. These cultures were exposed to non-resident macrophages. In untreated control cultures, there was no indication of myelin removal by the added macrophages. DRG were exposed to enzymatically generated oxygen radicals using the xanthin/xanthin oxidase or the glucose/glucose oxidase system. Assessment of Schwann cell viability and ultrastructural morphology revealed different patterns of cell cytotoxicity and morphological changes in different experiments. High concentrations caused complete tissue necrosis of the DRG, while low concentrations did not affect either cell viability or ultrastructural morphology. Under intermediate experimental conditions, oxygen radicals caused non-lethal Schwann cell damage leading to Schwann cell retraction and myelin sheath rejection. Myelin lamellae were disrupted and decompacted. These changes were followed by a selective macrophage attack on myelin sheaths, resulting in demyelination. Axons, Schwann cells and sensory ganglion cells survived this attack. The specificity of the oxygen radical effects was tested in experiments using the oxygen radical scavengers catalase and superoxide dismutase. Catalase prevented the described effects on cell morphology and subsequently blocked demyelination by non-resident macrophages.

Animals↗
PubMed1996

Development of an L6 myoblast in vitro model of moniliformin toxicosis.

L6 myoblasts were used as an in vitro model to investigate the role of moniliformin and its interaction with monensin in turkey knockdown syndrome and sudden death syndromes in poultry. Cell viability and microscopic and ultrastructural alterations noted in L6 myoblasts cultured in the presence of moniliformin (0.0-0.3 microgram/microliter) were compared to those observed in parallel cultures also containing one of the following compounds: selenium (0-0.004 ng/microliter), thiamine (0-0.3 microgram/microliter), or pyruvate (0-0.46 microgram/microliter). Marked dilation of the RER, membranous whorls, glycogen deposition, membrane-bound cytoplasmic inclusions and necrosis were observed in myoblasts exposed to 0.03-0.30 microgram moniliformin/microliter medium. Supplementation of medium with thiamine and pyruvate, or selenium, provided significant protection to cells exposed to 0.0-0.3 microgram/microliter or 0.0-0.15 microgram moniliformin/microliter, respectively. Dose-dependent differences in protein and ATP production were not detected. Myoblasts grown in medium containing 0-0.15 microgram moniliformin/microliter and 7.5-50.0 microM A23187, beauvericin or monensin had degrees of cytotoxicity similar to parallel cultures receiving only an ionophore. L6 myoblasts were a useful model of moniliformin toxicosis. The findings of this study suggest cytotoxicity due to moniliformin in L6 myoblasts may be due in part to oxidative damage and altered pyruvate metabolism, and that moniliformin does not predispose myoblasts to ionophore toxicosis. This study supports the results of in vivo investigations in poultry that moniliformin and monensin do not act synergistically to induce knockdown or monensin toxicosis.

Adenosine Triphosphate↗
PubMed2005

Essential role of extracellular matrix (ECM) overlay in establishing the functional integrity of primary neonatal rat Sertoli cell/gonocyte co-cultures: an improved in vitro model for assessment of male reproductive toxicity.

The development of in vitro models for testicular toxicity may provide important tools for investigating specific mechanisms of toxicity in the testis. Although various systems have been reported, their application in toxicological studies has been limited by the poor ability to replicate the complex biochemical, molecular, and functional interactions observed in the testis. In the present study, we evaluated a significantly improved Sertoli cell/gonocyte co-culture (SGC) system that employs a 3-dimensional extracellular matrix Matrigel (ECM) applied as an overlay instead of a substratum. We explored the dose- and time-dependent effects of the addition of such an ECM overlay on cytoskeletal and morphological changes in the SGC system, and the resulting effects on cellular integrity. Furthermore, we correlated the latter effects with the ECM-dependent modulation of stress and survival signaling pathways and, most critically, the expression levels of the spermatogonia-specific protein, c-Kit. Finally, we applied this co-culture system to investigate the dose- and time-dependent effects on the morphology and induction of apoptosis of cadmium. We observed that the dose-dependent addition of an ECM overlay led to an enhanced attachment of Sertoli cells and facilitated the establishment of SGC communication and cytoskeletal structure, with a dramatic improvement in cell viability. The latter was consistent with the observed dose- and time-dependent modulation of both stress signaling pathways (SAPK/JNK) and survival signaling pathways (ERK and AKT) in the presence of the ECM overlay. Furthermore, the dose-dependent stabilization of c-Kit protein expression confirmed the functional integrity of this co-culture system. We conclude that this modified SGC system will provide investigators with a simple, efficient, and highly reproducible alternative in the screen for testicular cell-specific cytotoxicity and the assessment of molecular mechanisms associated with both normal development and reproductive toxicity induced by environmental toxicants.

Actins↗
PubMed2005

Influence of growth hormone on maintenance of capillary-like structures in an in vitro model of stromal vascular tissue--results from morphometric analysis.

The in vitro development of a vascular stroma might be a solution for the engineering of vascularized tissues, however, in vitro stability of capillary-like structures is limited. In order to test the influence on maintenance of capillary-like structures, human growth hormone (hGH) was added in concentrations of 0.5, 5, 50, and 500 ng/mL in an in vitro model of stromal vascular tissue. The angiogenic response and maintenance of capillary-like structures were analyzed by means of confocal laser scanning microscopy (CLSM) and image analysis after 8, 16, and 32 days of culture. The highest angiogenic response was observed with a concentration of 50 ng/mL hGH. With the addition of 50 and 500 ng/mL, the length of capillary-like structures could be maintained on high levels up to the 32nd day of culture, whereas with 5 ng/mL values dropped to the level of the control group. The proposed technique of analysis allows quantification of capillary-like network formation and might be useful for tissue engineering applications.

Capillaries↗